The research described in this application continues to focus on the cellular and molecular biology of three enzymes of mammalian amino acid and organic acid metabolism whose inherited deficiencies lead to clinically significant metabolic disease in man: ornithine transcarbamylase (OTC), propionyl CoA carboxylase (PCC), and methylmalonyl CoA mutase (MUT). The experiments proposed are designed 1) to further delineate the pathway by which the cytoplasmic precursors of these mitochondrial matrix enzymes are targeted, translocated, processed and assembled, with specific emphasis on the role of cytoplasmic factors, the structure of the translocation complex, and the participation of heat-shook proteins or chaperonins in mitochondrial protein assembly: 2) to purify, characterize, clone, and sequence the two mitochondrial protease responsible for processing the OTC precursor; 3) to clarify the role of biotinylation in the transport and processing of alphaPCC; and 4) to apply the information, techniques, and reagents developed to a better understanding of the molecular bases of inherited deficiencies of these enzymes and to the improvement of diagnostic methods for these disease in vivo and in utero. For many of the studies planned, the general experimental design will involve the reconstitution in vitro of various aspects of rat liver mitochondrial protein import and processing. Specific techniques employed will include: isolation of intact, functional rat liver mitochondria and fractions thereof: protein purification by both conventional and affinity techniques; antibody generation and immunoprecipitation; preparation of mRNA and genomic DNA from patient material; Southern Northern, and Western blotting: and polymerase chain reaction amplification, cloning, manipulation and sequencing of cDNA and genomic DNA. These studies will provide new information on the fundamental processes of intracellular protein sorting and transport and should improve understanding of the role of this system and its components in both normal homeostasis and the pathophysiology off human genetic disease.